Varicella zoster virus (VZV), the cause of varicella (chickenpox) and zoster (shingles), is highly infectious but spreads only by cell-to-cell contact in vitro. Cultured cells do not release infectious virions because newly enveloped VZV follows the itinerary" " of cation independent mannose 6-phosphate receptors (MPRcis) to late endosomes, which are acidic and degrade virions before exocytosis. Final envelopment of VZV occurs in the trans-Golgi network (TGN). Viral glycoproteins (gps) segregate from cellular proteins within "C"-shaped TGN cisternae, so that opposite faces form (1) the envelope of a virion within (ii) a transport vesicle that contains MPRcis. When gI, or its C-terminal, is deleted, segregation and envelopment fail; viral gps and a tegument coat spreads around the entire circumference of TGN sacs, which adhere. Presence in the TGN of all gps is required for envelopment. Some gps ("navigators"), e.g. gE/gI contain TGN targeting signals, while others, e.g. gE, gH/gL ("passengers ') reach the TGN because they interact with "navigators". Tegument is postulated to bind to gp endodomains, enabling co-expressed gps to target tegument proteins, such as ORF10p, to the TGN. The current proposal will identify the gI domain(s) that are critical in the segregation of cellular and viral gps/tegument in TGN cisternae. Investigations will include determining: (1) the relative roles played by gE and gI in the delivery of "passengers" to the TGN; (2) the importance of endocytosis and endocytosis signals in the primary structures of gI, gE and the gE:gI complex in envelopment and in vitro spread of infection; (3) the roles played in gI and gE internalization of caveolin-containing vesicles and clathrin-coated pits. (4) whether gK contains a TGN targeting signal, or whether gK and gC are sorted to the TGN in complexes with gI and/or gE as "navigators". To test the hypotheses that VZV entry into target cells and diversion to late endosomes, are both MPRci-dependent, a line of MPRci-deficient human cells (MPR-KO) was generated. MPR-KO cells resist infection with cell-free, but not cell associated VZV and, once infected, they do not divert VZV to endosomes but secrete infectious VZV. MPR-KO cells will be used to evaluate the roles played by specific domains of VZV in viral envelopment. New studies will confirm that the resistance of MPR-KO cells to infection and the shedding of infectious virus is due to the knockdown of MPRcis; a cell line will be generated that expresses a dominant negative form of MPRci (the soluble ectodomain) to compete with endogenous MPRci for binding to the envelope gps of VZV. Understanding its gps is thus critical for understanding VZV. Knowledge of the actions of the gps may enable novel means to be developed to counter them and prevent the spread of VZV to new hosts or within hosts following infection. It may also make it possible to develop a safer and more efficacious vaccine.